Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

CRISPR-Cas9 knockdown of ESR1 in preoptic GABA-kisspeptin neurons suppresses the preovulatory surge and estrous cycles in female mice.

Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the preovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60-70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the regions of the rostral periventricular area of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being mice with bilateral ESR1 deletion in RP3V GABA neurons having reduced cFos expression in gonadotropin-releasing hormone (GnRH) neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near-complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile luteinizing hormone (LH) secretion. These observations demonstrate that ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.

Mechanism of Arcuate Kisspeptin Neuron Synchronization in Acute Brain Slices From Female Mice.

The mechanism by which arcuate kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to drive pulsatile hormone secretion remains unclear in females. In order to study spontaneous synchronization within the ARNKISS neuron network, acute brain slices were prepared from adult female Kiss1-GCaMP6 mice. Analysis of both spontaneous synchronizations and those driven by high frequency stimulation of individual ARNKISS neurons revealed that the network exhibits semi-random emergent excitation dependent upon glutamate signaling through AMPA receptors. No role for NMDA receptors was identified. In contrast to male mice, ongoing tachykinin receptor tone within the slice operated to promote spontaneous synchronizations in females. As previously observed in males, we found that ongoing dynorphin transmission in the slice did not contribute to synchronization events. These observations indicate that a very similar AMPA receptor-dependent mechanism underlies ARNKISS neuron synchronizations in the female mouse supporting the "glutamate two-transition" model for kisspeptin neuron synchronization. However, a potentially important sex difference appears to exist with a more prominent facilitatory role for tachykinin transmission in the female.

Unexpected Plasma Gonadal Steroid and Prolactin Levels Across the Mouse Estrous Cycle.

Despite the importance of the mouse in biomedical research, the levels of circulating gonadal steroids across the estrous cycle are not established with any temporal precision. Using liquid chromatography-mass spectrometry, now considered the gold standard for steroid hormone analysis, we aimed to generate a detailed profile of gonadal steroid levels across the estrous cycle of C57BL/6J mice. For reference, luteinizing hormone (LH) and prolactin concentrations were measured in the same samples by sandwich enzyme-linked immunosorbent assay. Terminal blood samples were collected at 8-hour intervals (10 Am, 6 Pm, 2 Am) throughout the 4 stages of the estrous cycle. As expected, the LH surge was detected at 6 Pm on proestrus with a mean (±SEM) concentration of 11 ± 3 ng/mL and occurred coincident with the peak in progesterone levels (22 ± 4 ng/mL). Surprisingly, estradiol concentrations peaked at 10 Am on diestrus (51 ± 8 pg/mL), with levels on proestrus 6 Pm reaching only two-thirds of this value (31 ± 5 pg/mL). We also observed a proestrus peak in prolactin concentrations (132.5 ± 17 ng/mL) that occurred earlier than expected at 2 Am. Estrone and androstenedione levels were often close to the limit of detection (LOD) and showed no consistent changes across the estrous cycle. Testosterone levels were rarely above the LOD (0.01 ng/mL). These observations provide the first detailed assessment of fluctuating gonadal steroid and reproductive hormone levels across the mouse estrous cycle and indicate that species differences exist between mice and other spontaneously ovulating species.

Mechanism of kisspeptin neuron synchronization for pulsatile hormone secretion in male mice.

The mechanism by which arcuate nucleus kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to generate pulsatile hormone secretion remains unknown. An acute brain slice preparation maintaining synchronized ARNKISS neuron burst firing was used alongside in vivo GCaMP GRIN lens microendoscope and fiber photometry imaging coupled with intra-ARN microinfusion. Studies in intact and gonadectomized male mice revealed that ARNKISS neuron synchronizations result from near-random emergent network activity within the population and that this was critically dependent on local glutamate-AMPA signaling. Whereas neurokinin B operated to potentiate glutamate-generated synchronizations, dynorphin-kappa opioid tone within the network served as a gate for synchronization initiation. These observations force a departure from the existing "KNDy hypothesis" for ARNKISS neuron synchronization. A "glutamate two-transition" mechanism is proposed to underlie synchronizations in this key hypothalamic central pattern generator driving mammalian fertility.

Robust GABAergic Regulation of the GnRH Neuron Distal Dendron.

The amino acid transmitter γ-aminobutyric acid (GABA) is suspected to play an important role in regulating the activity of the gonadotropin-releasing hormone (GnRH) neurons controlling fertility. Rodent GnRH neurons have a novel dendritic compartment termed the "distal dendron" through which action potentials pass to the axon terminals and where inputs from the kisspeptin pulse generator drive pulsatile GnRH secretion. Combining Gnrh1-Cre mice with the Cre-dependent calcium sensor GCaMP6 and confocal imaging of acute brain slices, we examined whether GABA regulated intracellular calcium concentrations ([Ca2+]) in the GnRH neuron distal dendron. Short puffs of GABA on the dendron evoked either a monophasic sustained suppression of [Ca2+] or a biphasic acute elevation in [Ca2+] followed by the sustained suppression. Application of muscimol to the dendron replicated the acute elevation in [Ca2+] while baclofen generated the sustained suppression. Robust GABAB receptor-mediated inhibition was observed in 80% to 100% of dendrons recorded from females across the estrous cycle and from approximately 70% of dendrons in males. In contrast, the GABAA receptor-mediated excitation was rare in males and varied across the estrous cycle, being most prominent at proestrus. The activation of GABAB receptors potently suppressed the stimulatory effect of kisspeptin on the dendron. These observations demonstrate that the great majority of GnRH neuron distal dendrons are regulated by GABAergic inputs in a sex- and estrous cycle-dependent manner, with robust GABAB receptor-mediated inhibition being the primary mode of signaling. This provides a new, kisspeptin-independent, pathway for the regulation of pulsatile and surge modes of GnRH secretion in the rodent.

Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice.

The mechanisms underlying the homeostatic estrogen negative feedback pathway central to mammalian fertility have remained unresolved. Direct measurement of gonadotropin-releasing hormone (GnRH) pulse generator activity in freely behaving mice with GCaMP photometry demonstrated striking estradiol-dependent plasticity in the frequency, duration, amplitude, and profile of pulse generator synchronization events. Mice with Cre-dependent deletion of ESR1 from all kisspeptin neurons exhibited pulse generator activity identical to that of ovariectomized wild-type mice. An in vivo CRISPR-Cas9 approach was used to knockdown ESR1 expression selectively in arcuate nucleus (ARN) kisspeptin neurons. Mice with >80% deletion of ESR1 in ARN kisspeptin neurons exhibited the ovariectomized pattern of GnRH pulse generator activity and high frequency LH pulses but with very low amplitude due to reduced responsiveness of the pituitary. Together, these studies demonstrate that estrogen utilizes ESR1 in ARN kisspeptin neurons to achieve estrogen negative feedback of the GnRH pulse generator in mice.

Neuroendocrine control of gonadotropin-releasing hormone: Pulsatile and surge modes of secretion.

The concept that different systems control episodic and surge secretion of gonadotropin-releasing hormone (GnRH) was well established by the time that GnRH was identified and formed the framework for studies of the physiological roles of GnRH, and later kisspeptin. Here, we focus on recent studies identifying the neural mechanisms underlying these two modes of secretion, with an emphasis on their core components. There is now compelling data that kisspeptin neurons in the arcuate nucleus that also contain neurokinin B (NKB) and dynorphin (i.e., KNDy cells) and their projections to GnRH dendrons constitute the GnRH pulse generator in mice and rats. There is also strong evidence for a similar role for KNDy neurons in sheep and goats, and weaker data in monkeys and humans. However, whether KNDy neurons act on GnRH dendrons and/or GnRH soma and dendrites that are found in the mediobasal hypothalamus (MBH) of these species remains unclear. The core components of the GnRH/luteinising hormone surge consist of an endocrine signal that initiates the process and a neural trigger that drives GnRH secretion during the surge. In all spontaneous ovulators, the core endocrine signal is a rise in estradiol secretion from the maturing follicle(s), with the site of estrogen positive feedback being the rostral periventricular kisspeptin neurons in rodents and neurons in the MBH of sheep and primates. There is considerable species variations in the neural trigger, with three major classes. First, in reflex ovulators, this trigger is initiated by coitus and carried to the hypothalamus by neural or vascular pathways. Second, in rodents, there is a time of day signal that originates in the suprachiasmatic nucleus and activates rostral periventricular kisspeptin neurons and GnRH soma and dendrites. Finally, in sheep nitric oxide-producing neurons in the ventromedial nucleus, KNDy neurons and rostral kisspeptin neurons all appear to participate in driving GnRH release during the surge.

Reformulation of PULSAR for Analysis of Pulsatile LH Secretion and a Revised Model of Estrogen-Negative Feedback in Mice.

The recent use of the tail-tip bleeding approach in mice has enabled researchers to generate detailed pulse and surge profiles of luteinizing hormone (LH) secretion in mice. However, the analysis of pulsatile LH secretion is piecemeal across the field with each laboratory using their own methodology. We have reformulated the once-popular PULSAR algorithm of Merriam and Wachter to operate on contemporary computer systems and provide downloadable and online pulse analysis platforms. As it is now possible to record the activity of the gonadotropin-releasing hormone pulse generator in freely behaving mice, we have been able to unambiguously define LH pulses in intact and gonadectomized male and female mice. These data sets were used to determine the appropriate PULSAR parameter sets for analyzing pulsatile LH secretion in the mouse. This was then used to establish an accurate model of estrogen negative feedback in the mouse. Intact and ovariectomized mice given Silastic capsules containing 1, 2, and 4 µg 17-ß-estradiol/20 g body weight were tail-tip bled at 6-min intervals, and the resultant LH profiles were analyzed with PULSAR. Only the 4 µg 17-ß-estradiol capsule treatment was found to return LH pulse amplitude and frequency to that of intact diestrous mice. Ultrasensitive mass spectrometry analysis showed that the 4 µg 17-ß-estradiol capsule generated circulating estradiol levels equivalent to that of diestrous mice. It is hoped that the reformulation of PULSAR and generation of a realistic model of estrogen-negative feedback will provide a platform for the more uniform assessment of pulsatile hormone secretion in mice.

The dendron and episodic neuropeptide release.

The unexpected observation that the long processes of gonadotrophin-releasing hormone (GnRH) neurons not only conducted action potentials, but also operated to integrate afferent information at their distal-most extent gave rise to the concept of a blended dendritic-axonal process termed the "dendron". The proximal dendrites of the GnRH neuron function in a conventional manner, receiving synaptic inputs and initiating action potentials that are critical for the surge mode of GnRH secretion. The distal dendrons are regulated by both classical synapses and volume transmission and likely operate using subthreshold electrotonic propagation into the nearby axon terminals in the median eminence. Evidence indicates that neural processing at the distal dendron is responsible for the pulsatile patterning of GnRH secretion. Although the dendron remains unique to the GnRH neuron, data show that it exists in both mice and rats and may be a common feature of mammalian species in which GnRH neuron cell bodies do not migrate into the basal hypothalamus. This review outlines the discovery and function of the dendron as a unique neuronal structure optimised to generate episodic neuronal output.

Morphological assessment of GABA and glutamate inputs to GnRH neurons in intact female mice using expansion microscopy.

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05-0.08 synapses µm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.

Impact of chronic variable stress on neuroendocrine hypothalamus and pituitary in male and female C57BL/6J mice.

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single-point blood measurements of basal prolactin, thyroid-stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt, Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up-regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS.

Indirect Suppression of Pulsatile LH Secretion by CRH Neurons in the Female Mouse.

Acute stress is a potent suppressor of pulsatile luteinizing hormone (LH) secretion, but the mechanisms through which corticotrophin-releasing hormone (CRH) neurons inhibit gonadotropin-releasing hormone (GnRH) release remain unclear. The activation of paraventricular nucleus (PVN) CRH neurons with Cre-dependent hM3Dq in Crh-Cre female mice resulted in the robust suppression of pulsatile LH secretion. Channelrhodopsin (ChR2)-assisted circuit mapping revealed that PVN CRH neuron projections existed around kisspeptin neurons in the arcuate nucleus (ARN) although many more fibers made close appositions with GnRH neuron distal dendrons in the ventral ARN. Acutely prepared brain slice electrophysiology experiments in GnRH- green fluorescent protein (GFP) mice showed a dose-dependent (30 and 300 nM CRH) activation of firing in ~20% of GnRH neurons in both intact diestrus and ovariectomized mice with inhibitory effects being uncommon (<8%). Confocal GCaMP6 imaging of GnRH neuron distal dendrons in acute para-horizontal brain slices from GnRH-Cre mice injected with Cre-dependent GCaMP6s adeno-associated viruses demonstrated no effects of 30 to 300 nM CRH on GnRH neuron dendron calcium concentrations. Electrophysiological recordings of ARN kisspeptin neurons in Crh-Cre,Kiss1-GFP mice revealed no effects of 30 -300 nM CRH on basal or neurokinin B-stimulated firing rate. Similarly, the optogenetic activation (2-20 Hz) of CRH nerve terminals in the ARN of Crh-Cre,Kiss1-GFP mice injected with Cre-dependent ChR2 had no effect on kisspeptin neuron firing. Together, these studies demonstrate that PVN CRH neurons potently suppress LH pulsatility but do not exert direct inhibitory control over GnRH neurons, at their cell body or dendron, or the ARN kisspeptin neuron pulse generator in the female mouse.

Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron.

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic 'KNDy' neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model.

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed "dendrons," and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

Different dendritic domains of the GnRH neuron underlie the pulse and surge modes of GnRH secretion in female mice.

The gonadotropin-releasing hormone (GnRH) neurons exhibit pulse and surge modes of activity to control fertility. They also exhibit an unusual bipolar morphology comprised of a classical soma-proximal dendritic zone and an elongated secretory process that can operate as both a dendrite and an axon, termed a 'dendron'. We show using expansion microscopy that the highest density of synaptic inputs to a GnRH neuron exists at its distal dendron. In vivo, selective chemogenetic inhibition of the GnRH neuron distal dendron abolishes the luteinizing hormone (LH) surge and markedly dampens LH pulses. In contrast, inhibitory chemogenetic and optogenetic strategies targeting the GnRH neuron soma-proximal dendritic zone abolish the LH surge but have no effect upon LH pulsatility. These observations indicate that electrical activity at the soma-proximal dendrites of the GnRH neuron is only essential for the LH surge while the distal dendron represents an autonomous zone where synaptic integration drives pulsatile GnRH secretion.

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse.

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin-releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic-sensing pro-opiomelanocortin and agouti-related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin-releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1-GFP and Kiss1-GCaMP6 mice, we investigated whether NPY and α-melanocyte-stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B-evoked firing of approximately one-half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B-evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α-melanocyte-stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.

A simple model of estrous cycle negative and positive feedback regulation of GnRH secretion.

The gonadal steroids estradiol and progesterone exert critical suppressive and stimulatory actions upon the brain to control gonadotropin-releasing hormone (GnRH) release that drives the estrous/menstrual cycle. A simple model for understanding these interactions is proposed in which the activity of the "GnRH pulse generator" is restrained by post-ovulation progesterone secretion to bring about the estrus/luteal phase slowing of pulsatile gonadotropin release, while the activity of the "GnRH surge generator" is primed by the rising follicular phase levels of estradiol to generate the pre-ovulatory surge. The physiological fluctuations in estradiol levels across the cycle are considered to clamp the GnRH pulse generator output at a constant level. Independent pulse and surge generator circuitries regulate the excitability of different compartments of the GnRH neuron. As such, GnRH secretion through the cycle is determined simply by the summed influence of the estradiol-clamped, progesterone-regulated pulse and estradiol-regulated surge generators on the GnRH neuron.

Neural Determinants of Pulsatile Luteinizing Hormone Secretion in Male Mice.

The gonadotrophin-releasing hormone (GnRH) pulse generator drives pulsatile luteinizing hormone (LH) secretion essential for fertility. However, the constraints within which the pulse generator operates to drive efficient LH pulsatility remain unclear. We used optogenetic activation of the arcuate nucleus kisspeptin neurons, recently identified as the GnRH pulse generator, to assess the efficiency of different pulse generator frequencies in driving pulsatile LH secretion in intact freely behaving male mice. Activating the pulse generator at 45-minute intervals generated LH pulses similar to those observed in intact male mice while 9-minute interval stimulation generated LH profiles indistinguishable from gonadectomized (GDX) male mice. However, more frequent activation of the pulse generator resulted in disordered LH secretion. Optogenetic experiments directly activating the distal projections of the GnRH neuron gave the exact same results, indicating the pituitary to be the locus of the high frequency decoding. To evaluate the state-dependent behavior of the pulse generator, the effects of high-frequency activation of the arcuate kisspeptin neurons were compared in GDX and intact mice. The same stimulus resulted in an overall inhibition of LH release in GDX mice but stimulation in intact males. These studies demonstrate that the GnRH pulse generator is the primary determinant of LH pulse profile and that a nonlinear relationship exists between pulse generator frequency and LH pulse frequency. This may underlie the ability of stimulatory inputs to the pulse generator to have opposite effects on LH secretion in intact and GDX animals.

Activation of a Classic Hunger Circuit Slows Luteinizing Hormone Pulsatility.

INTRODUCTION: The central regulation of fertility is carefully coordinated with energy homeostasis, and infertility is frequently the outcome of energy imbalance. Neurons in the hypothalamus expressing neuropeptide Y and agouti-related peptide (NPY/AgRP neurons) are strongly implicated in linking metabolic cues with fertility regulation. OBJECTIVE: We aimed here to determine the impact of selectively activating NPY/AgRP neurons, critical regulators of metabolism, on the activity of luteinizing hormone (LH) pulse generation. METHODS: We employed a suite of in vivo optogenetic and chemogenetic approaches with serial measurements of LH to determine the impact of selectively activating NPY/AgRP neurons on dynamic LH secretion. In addition, electrophysiological studies in ex vivo brain slices were employed to ascertain the functional impact of activating NPY/AgRP neurons on gonadotropin-releasing hormone (GnRH) neurons. RESULTS: Selective activation of NPY/AgRP neurons significantly decreased post-castration LH secretion. This was observed in males and females, as well as in prenatally androgenized females that recapitulate the persistently elevated LH pulse frequency characteristic of polycystic ovary syndrome (PCOS). Reduced LH pulse frequency was also observed when optogenetic stimulation was restricted to NPY/AgRP fiber projections surrounding GnRH neuron cell bodies in the rostral preoptic area. However, electrophysiological studies in ex vivo brain slices indicated these effects were likely to be indirect. CONCLUSIONS: These data demonstrate the ability of NPY/AgRP neuronal signaling to modulate and, specifically, reduce GnRH/LH pulse generation. The findings suggest a mechanism by which increased activity of this hunger circuit, in response to negative energy balance, mediates impaired fertility in otherwise reproductively fit states, and highlight a potential mechanism to slow LH pulsatility in female infertility disorders, such as PCOS, that are associated with hyperactive LH secretion.